A type of solid phase assay devices comprises a plate-shaped flow matrix of bibulous material, usually a membrane strip, such as of cellulose nitrate or glass fiber, in which liquid can be transported laterally (i.e. in the plane of the strip) by capillary forces in the membrane. The membrane usually has a sample application zone, and a detection zone downstream of the sample application zone. In the detection zone, usually a capturing reagent for the analyte is immobilized. To conduct an assay, the application zone is contacted with the liquid sample to be assayed for the analyte of interest. The device is maintained under conditions sufficient to allow capillary action of liquid to transport the analyte of interest, if present in the sample, through the membrane strip to the detection zone where the analyte is captured. The capillary liquid flow is usually insured by an absorbing pad or the like at the downstream end of the strip. A detection reagent, usually labelled, is then added upstream of the detection zone and interacts with captured analyte in the detection zone, and the amount of captured analyte is measured. Often, the detection reagent is pre-deposited in or on the membrane strip, e.g. in the form of diffusively movable particles containing fluorophoric or chromogenic groups, either upstream of the sample application zone or between the sample application zone and the detection zone.
Since it takes some time for the sample and the assay liquids to be transported through the detection zone such that the result of the assay can be read, it has been proposed to provide a timing control, such as a “timer” substance or substance combination on the strip which indicates when flow through the flow matrix has occurred or that enough time has elapsed from the time that a fluid sample was applied to the membrane strip for the reading to give a correct value.
EP-A-915 336 discloses a chromatographic assay device wherein the chromatographic medium includes a resolubilizable visible dye in an area between the detection zone and the end of the chromatographic medium, e.g. applied in the absorbing pad. During the performance of the assay, the dye in the dye area is resolubilized and migrates from the dye area to a dye viewing area which gives a visual indication that flow through the chromatographic medium has occurred, such that the assay result can be read and interpreted. The resolubilizable dye may be bound to a first member of a specific binding pair, and a second member of the specific binding pair may be immobilized in the dye viewing area to capture the dye therein. The timing control of EP-A-915 336, however, only indicates that flow through the flow matrix has taken place and does not provide for any adjustment of the time elapsed from the start of the assay until the colour is visible in the viewing area.
This shortcoming is to some extent overcome by the chemical timer disclosed in EP-A-826 777. The chemical timer, which is used in a visible test strip for measuring the concentration of an analyte in a biological fluid that is applied to the strip, measures a predetermined interval chemically and comprises a dry coating of (i) a coloured indicator composition, (ii) a reagent that, when hydrated, is capable of reacting with glucose to change the colour of the indicator, (iii) an inhibitor to inhibit the change in colour of the indicator, and (iv) glucose, in which the inhibitor and glucose concentrations in the dry coating are selected so that the glucose, over a predetermined time after the biological fluid sample is applied to the strip, reacts with the reagent to change the colour of the indicator. When a sample is applied to the strip, hydration of the timer segment composition permits the colour-forming reaction to proceed. The time it takes for the timer segment to change colour is determined by the temperature and by characteristics of the testing reagent, particularly the inhibitor concentration, the amount of glucose, and the hydration and oxygen diffusion rates. The timer also serves as a quality control function, by making it apparent when a test strip has been contaminated by exposure to moisture. Migration of indicators having such a tendency may be prevented by including an ion pairing agent in the matrix.
While the time to colour-change of the chemical timer described in EP-A-826 777 may be varied, this is not readily done, requiring inter alia a different composition of the timer segment for each desired colour-change time. Since the time when the assay result may be reliably read varies between different assay formats depending inter alia on the number of the assay liquids used, there is therefore a need for a test strip having a more flexible timer that can easily be adjusted to a desired indication time to suit the requirements of a particular assay.